Herpes virus complementing cell line

ABSTRACT

The present invention is directed to a cell line capable of supporting replication of a growth-defective Herpes Simplex Virus strain; specifically a replication-defective HSV-2 double mutant. Particularly disclosed is a cell line that expresses the ICP8 protein and the UL5 protein of Herpes Simplex Virus. This cell line is useful to propagate a replication-defective HSV-2 vaccine strain that contains mutations and/or deletions in the ICP8 and UL5 genes.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. provisional application No.60/196,801, filed Apr. 13, 2000.

FIELD OF THE INVENTION

The present invention is in the fields of cellular and molecularbiology. Specifically, The present invention is directed to a cell lineuseful for the growth of a mutant strain of Herpesvirus.

BACKGROUND OF THE INVENTION

Herpesviridae is a large family of enveloped linear dsDNA-containinganimal viruses. Herpesviruses are morphologically similar. The virion(˜120-200 nm diam.) contains a core (DNA wound around a central proteinstructure) within an icosahedral capsid (˜100-110 nm diam.) comprising12 pentameric and 150 hexameric capsomers. The viron is enclosed by alipoprotein envelope bearing surface projections. The linear dsDNAgenome characteristically contains repeated terminal and/or internalsequences.

Herpesviruses have been isolated from a wide range of animals, includingmammals, birds, reptiles, amphibians, and fish. Many herpesviruses causedisease in their primary host(s), and may remain latent within thetissue of the host, often for life. Virus transmission commonly occursby direct contact of mucosal surfaces. Some herpesviruses can betransmitted via body fluids (e.g., milk; via the placenta; etc).

Herpes simplex virus (HSV) type 1 or 2 is a causative agent for seriousinfections in humans. Herpes simplex diseases are characterized by theformation of thin-walled vesicles, which ulcerate, crust and heal; thevesicles occur, often in clusters, on skin and/or mucous membranes.Transmission occurs as a result of close physical contact; e.g., sexualcontact, kissing, close contact sports such as wrestling (Herpesgladiatorum). HSV incubation periods range from 2-12 (average 6) days.The disease state varies from subclinical to severe, and is occasionallyfatal. HSV can remain latent in nerve cells near the site of infection.Reactivation may occur spontaneously or in response to other infections(e.g., stress, immunosuppression). In neonates and immunodeficientindividuals, HSV may become-disseminated; often affecting the liver,adrenal glands, brain, etc.

HSV-2 is associated with genital, and hence neonatal, infections (Herpesgenitalis), and is a disease of significant morbidity in infectedindividuals (Whitley, 1996). In addition to the conditions describedabove, other symptoms may include e.g. fever, dysuria, pain, andmalaise. In women, the cervix is often the main site of genitalinfection (herpetic cervicitis). HSV-2 infection in women is associatedwith an increased risk of abortion and of cervical cancer. Individualswith active HSV-2 have an increased risk of acquiring HIV if exposed tothe virus (Augenbraun and McCormack).

Neonatal herpes is usually acquired (during birth) from a motherinfected with HSV-2. Fatality rates may be 50% or more in untreatedcases. Surviving infants commonly show neurological and/or ocularsecondary disorders.

Clinical diagnosis of HSV-2 infection is established by microscopicexamination of lesion samples, or biopsies from e.g. skin, brain orliver for multinucleate giant cells with eosinophilic intranuclearinclusion bodies, or by various immunofluorescence techniques (e.g.,ELISA).

A number of antiviral agents (e.g. vidarabine, acyclovir, IDU andtrifluorothymidine) have activity against HSV and may be effective insome cases (e.g. vidarabine is used against HSV encephalitis). Thesedrugs are not generally effective in preventing recurrence ortransmission, however. There remains an unmet need for an effectivevaccine against HSV-2 to induce protective immunity and to prevent or toreduce primary infection and, ideally, to reduce recurrent disease andtransmission.

Numerous approaches have been attempted to obtain immunization againstHSV infection (e.g., glycoprotein subunits, inactivated virus,attenuated virus, and various HSV antigens. See Krause and Straus, 1999;Bernstein and Stanberry, 1999; and Stanberry, 1998). These approacheshave shown little to no effectiveness, however (e.g., HSV glycoproteinsubunit vaccines, Corey et al., 1999 and Straus et al, 1997; attenuatedHSV, Cadoz, et al., 1992).

The use of replication-defective mutant viral strains is a promisingavenue of induced immunization against HSV in animal models (Boursnellet al., 1997; Da Costa et al., 1997; Farrell et al., 1994; McLean etal., 1994; McLean, 1996; Morrison and Knipe, 1994; Morrison, Da Costa,and Knipe, 1998; Nguyen et al., 1992; and Stanberry, 1999). Currentstudies, however, use single mutant viruses, which carry with it thethreat of back mutation (reversion to a virulent wild type). Standardvaccine design typically utilizes strains with two or more(non-reverting) mutations to increase safety of the vaccine (Curtiss etal., 1994).

In an effort to develop a live mutant virus vaccine, while reducing therisk of reversion, Da Costa et al. have developed a double deletionmutant HSV-2 strain. This strain lacks two genes essential for DNAreplication, thus rendering the mutant incapable of DNA synthesis andviral replication. This double deletion mutant virus strain fails toform plaques or to give any detectable single cycle yields in normalmonkey or human cells, yet it is capable of eliciting an immune response(i.e., it functions as an effective immunogen). This double deletionmutant HSV-2 strain induces antibody titers in mice equivalent to thoseinduced by single deletion mutant viruses (Da Costa et al., manuscriptsubmitted).

Because this double deletion mutant HSV-2 strain is replicationdefective, the replication gene product components it lacks must beprovided. There is a need, therefore, for a cell line capable ofcomplementing this double deletion mutant HSV-2 strain, enabling thepropagation of the strain, thus providing vaccine production levelgrowth stock of the mutant strain.

SUMMARY OF THE INVENTION

The present invention is directed to a cell line capable of supportingreplication of a growth-defective Herpes Simplex Virus strain;specifically a replication-defective HSV-2 double mutant. Thiscomplementing cell line provides a means to propagate a growth defectivestrain of HSV useful as, for example, stock immunogen for vaccineproduction.

In one preferred embodiment, the present invention is directed to a cellline that expresses the ICP8 protein and the UL5 protein of HerpesSimplex Virus. This cell line is useful to propagate areplication-defective HSV-2 vaccine strain that contains mutationsand/or deletions in the ICP8 and UL5 genes. Most preferably, thecomplementing cell line exhibits the characteristics of thecomplementing cell line as deposited with the American Type Cell Culture(ATCC) and assigned the Patent Deposit Designation PTA-2403.

In another embodiment of the invention the UL5/ICP8 expressing cell linecontains an ICP8/UL5-defective HSV-2 strain.

Another embodiment of the present invention is directed to a method forproducing an ICP8/UL5-defective HSV-2 strain by propagating the mutantvirus using the UL5/ICP8 expressing cell line, and harvesting the virusresulting from the cell culture.

In one aspect of the present invention, the complementing cell linereduces the possibility of reversion of the defective virus to its wildtype form during replication in the complementing cell line. This isachieved by assuring the complementing cell line contains minimalsequence homology with the replication-defective HSV strain to avoidgenetic recombination and to produce only defective virus. This aspectis of added benefit because a complementing cell line with little or nosequence homology with a mutant HSV strain provides further safetyduring growth of the virus.

DETAILED DESCRIPTION OF THE INVENTION

Vero Cells (African Green Monkey Kidney Cells, ATCC #81-CCL) were usedto create a cell line that expresses the UL5 and ICP8 proteins of HerpesSimplex Virus. It is understood, however that any of a variety ofsuitable cell lines may be used in the practice of this invention.

As an initial step, suitable expression vectors for each of thedefective viral genes are created by any of a variety of techniquesknown in the art and using any of a number of suitable vectors.

Then, a cell line capable of expressing one of the two defective viralgenes is created, using any of a variety of genetic engineeringtechniques known in the art For example, an ICP8 expressing cell linemay be generated by transfecting Vero cells with an HSV-1 ICP8expression plasmid. A modified calcium phosphate method of transfectionmay be used to create the ICP8 expressing cell line. As describedherein, the ICP8 expression plasmid, pRC/CMV-ICP8-A1(RA1) may be used inthe construction of this cell line.

Finally, the cell line capable of expressing the first of the twodefective viral genes is manipulated to express the second of the twodefective viral genes. For example, an ICP8 expressing cell line may befurther transfected with a UL5 expression plasmid using, for example, anelectroporation method of transfection. As described herein, the UL5expression plasmid, p70-4 is useful in this transfection.

An ICP8/UL5 expressing cell line is selected on the basis of its abilityto support the replication of both an ICP8 mutant HSV-2 strain, and aUL5 mutant HSV-2 strain.

Many component steps of the present invention may be performed byalternative, yet functionally equivalent, biochemical or geneticengineering techniques known in the art without altering the inventivenature of the present disclosure. For example, methods for generatingrecombinant nucleic acids, vector construction, host celltransformation, polypeptide expression systems, and phage display usefulin the practice of this invention can involve a wide variety of moderngenetic engineering techniques, tools, and biological sources that arewell known in the art and routinely practiced by those skilled in theart. Exemplary techniques and methods are described in detail herein byway of preferred example, but are not limiting to the practice of theinvention. The present invention incorporates by reference in theirentirety techniques and supplies well known in the field of molecularbiology, including, but not limited to, techniques and suppliesdescribed in the following publications:

-   -   Ausubel, F. M. et al. eds., Short Protocols In Molecular Biology        (4th Ed. 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X).    -   Freshney, R. I. Culture of Animal Cells (1987) Alan R. Liss,        Inc.    -   Old, R. W. & S. B. Primrose, Principles of Gene Manipulation: An        Introduction To Genetic Engineering (3d Ed. 1985) Blackwell        Scientific Publications, Boston. Studies in Microbiology;        V.2:409 pp. (ISBN 0-632-01318-4).    -   Sambrook, J. et al. eds., Molecular Cloning: A Laboratory Manual        (2d Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols.        1-3. (ISBN 0-87969-309-6).    -   Winnacker, E. L. From Genes To Clones: Introduction To Gene        Technology (1987) VCH Publishers, NY (translated by Horst        Ibelgaufts). 634 pp. (ISBN 0-89573-6144).

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the compositions and methodsof the invention described herein are obvious and may be made withoutdeparting from the scope of the invention or the embodiments disclosedherein. Having now described the present invention in detail, the samewill be more clearly understood by reference to the following examples,which are included for purposes of illustration only and are notintended to be limiting of the invention.

Each of the publications mentioned herein is incorporated by reference.

EXAMPLE 1 Preparation of Reagents and Solutions Penicillin/StreptomycinSolution, 200×

200× Penicillin/Streptomycin was prepared as follows: A 0.9% NaClsolution was prepared by dissolving 0.9 g of NaCl in 100 ml of steriledeionized water. 200× Penicillin/Streptomycin solutions were prepared byseparately dissolving the contents of two bottles ofpenicillin/streptomycin base (Sigma, #P-3539, 5 g) in 50 ml of the 0.9%NaCl solution. The two 50 ml Penicillin/Streptomycin solutions werecombined and filter sterilized using 0.2 μM (100 ml capacity) filterunits. The 200× Penicillin/Streptomycin solutions were divided into 10ml aliquots and dispensed into sterile test tubes (15 ml capacity). Thesolutions were stored at −20° C. and thawed at room temperature prior touse.

Larger laboratory stock of 200× Penicillin/Streptomycin was prepared asfollows: Two 500 ml aliquots of PBS were filter sterilized usingseparate 0.2 μM filter units (500 ml capacity) prior to use. 200×Penicillin/Streptomycin solutions were prepared by separately dissolvingthe contents of two bottles of penicillin/streptomycin base (Sigma,#P-3539, 5 g) in 50 ml of sterile phosphate buffered saline, pH 7.4(PBS). The two 50 ml Penicillin/Streptomycin solutions were combined andfilter sterilized using 0.2 μM (100 ml capacity) filter units. The 200×Penicillin/Streptomycin solutions were divided into 10 ml aliquots anddispensed into sterile test tubes (15 ml capacity). The solutions werestored at −20° C. and thawed at room temperature prior to use.

Gentamicin (G418) Solution, 100 mg/ml

The microbial potency (active G418) for the lot of material was obtainedfrom the supplier's information provided with the material. The amountof diluent required to prepare a solution having a potency of 100 mg/mlactive G418 was determined as follows:

-   -   Microbial potency=740 μg/mg    -   740 μg/mg×5000 mg (contents of bottle)=3700 mg active G418    -   (3700 mg active G418)/(×ml of diluent)=100 mg active G418 (final        concentration)/(1 ml)    -   ×(ml of diluent)=37 ml DMEM

12 ml of Dulbecco's Modified Eagles Medium (DMEM) was added to 5 g ofG418 and dissolved by repeat pipetting. An additional 12 ml of DMEM wasadded to dissolve any remaining G418 powder. The final volume of theG418 solution was adjusted to 37 ml with DMEM. The solution was mixed byinversion and dispensed into sterile test tubes in 5 ml aliquots. TheG418 solution aliquots were stored at −20° C. and thawed at roomtemperature prior to use.

Fetal Bovine Serum, Heat Inactivated

Fetal bovine serum used as a cell culture media additive was heatinactivated. Bottles containing 100 ml of fetal bovine serum were storedat −20° C. Prior to use, a bottle of serum was placed in a 37° C. waterbath until the contents of the bottle had thawed. The bottle was thentransferred into a 56° C. water bath for 30 minutes. The heatinactivated serum was then used to prepare cell culture media.

Cell Culture Media:

V Media

The cell culture media designated “V” Media was prepared by adding thefollowing to a bottle containing 500 ml of DMEM: 55 ml of fetal bovineserum, heat inactivated; 2.8 ml 200× Penicillin/Streptomycin solution.The contents of the bottle were mixed by swirling the bottle. Theprepared media was stored at 4° C. between uses.

G Media

The cell culture media designated “G” Media was prepared by adding thefollowing to a bottle containing 500 ml of DMEM: 55 ml of fetal bovineserum, heat inactivated; 2.8 ml 200× Penicillin/Streptomycin solution;2.8 ml of 100 mg/ml G418 solution. The contents of the bottle were mixedby swirling the bottle. The prepared media was stored at 4° C. betweenuses.

S2 Media

The cell culture media designated “S2” Media was prepared by adding thefollowing to a bottle containing 500 ml of DMEM: 55 ml of fetal bovineserum, heat inactivated; 2.8 ml 200× Penicillin/Streptomycin solution;1.1 ml of 100 mg/ml G418 solution. The contents of the bottle were mixedby swirling the bottle. The prepared media was stored at 4° C. betweenuses.

S2Z Media

The cell culture media designated “S2Z” Media was prepared by adding thefollowing to a bottle containing 450 ml of DMEM (50 ml of DMEM removedfrom bottle containing 500 ml): 50 ml of fetal bovine serum, heatinactivated; 2.5 ml 200× Penicillin/Streptomycin solution; 1.0 ml of 100mg/ml G418 solution; 2.5 ml of 100 mg/ml Zeocin solution. The contentsof the bottle were mixed by swirling the bottle. The bottle containingthe prepared media was wrapped in aluminum foil and stored at 4° C.between uses.

Freezing Media

Freezing media was prepared by mixing equal volumes of DMEM with heatinactivated fetal bovine serum.

Transfections Solutions:

2× HBS (Hepes Buffered Saline)

2× HBS was prepared immediately prior to use by dissolving Hepes salt,NaCl and Na₂HPO₄ in 100 ml of sterile, distilled and deionized water asfollows:

Final Concentration Hepes Salt (FW = 260.3 g)  1.3 g   50 mM NaCl (FW =58.4 g)  1.6 g  280 mM Na₂HPO₄ (FW = 141.96) 0.02 g  1.5 mMThe 2× HBS solution was filter sterilized using a 0.2 μM filter unitprior to use.

1× HBS (Hepes Buffered Saline)

1× HBS was prepared immediately prior to use by dissolving Hepes salt,NaCl and Na₂HPO₄ in 100 ml of sterile, distilled and deionized water asfollows:

Final Concentration Hepes Salt (FW = 260.3 g) 0.65 g  25 mM NaCl (FW =58.4 g)  0.8 g 140 mM Na₂HPO₄ (FW = 141.96) 0.01 g 0.75 mM The 1× HBS solution was filter sterilized using a 0.2 μm filter unitprior to use.

2M CaCl₂

2M CaCl₂ was prepared immediately prior to use by dissolving 2.94 g ofCaCl₂ (FW=147.02) in 10 ml of sterile, distilled and deionized water.The 2M CaCl₂ solution was filter sterilized using a 0.2 μm filter unitprior to use.

EXAMPLE 2 Construction of an ICP8 Expression Vector:PRC/CMV-ICP8-A1(RA1)

The open reading frame (ORF) of the UL29 gene was inserted into thecommercial vector, pRc/CMV (Invitrogen Corporation, Carlsbad, Calif.,)in order to construct a plasmid that could express the protein, ICP8.

The UL29-ORF was constructed using polymerase chain reaction (PCR)amplification techniques known in the art. PCR primers were designed toallow amplification of the ORF of the UL29 gene only. Unique restrictionsites were engineered into the 5′ ends of the primers for directionalcloning of the PCR product into the commercial vector.

After amplifying the ORF, the blunt ended PCR product was cloned intothe EcoRV site of pBluescript II SK ASK) to generate pSK/UL29-ORF. TheUL29-ORF was subsequently cut out from pSK/UL29-ORF with HindII andXbaI, and cloned into the multiple cloning site of pRc/CMV to generatethe ICP8 expression plasmid.

The ICP8 expression plasmid contains the CMV immediate earlyenhancer/promoter, the open reading frame (ORF) from the UL29 gene, andthe bovine growth hormone polyA signal. The plasmid also contains theampicillin resistance gene for preparation of the plasmid in bacteria,and the neomycin resistance gene for selection of stable eukaryotic celllines.

This ICP8 expression plasmid was designated pRC/CMV-ICP8-A1(RA1)

EXAMPLE 3 Construction of a UL5 Expression Vector: P70-4

The open reading frame of the ULS gene from Herpes Simplex virus type 2(HSV-2) was amplified using PCR techniques. A plasmid containing the UL5gene cloned from HSV-2 was used as a template for the PCR amplification.

Plasmid pEH49 contains a 7.6 Kb EcoRI-HindIII fragment of HSV 2 DNAcontaining the UL5 gene. A 3327 bp KpnI-MluI sub fragment from pEH49 wascloned into the vector PSL1190, resulting in plasmid pUL5 (obtained fromXavier Da Costa, Harvard Medical School).

The HSV-2 DNA present in pUL5 was sequenced and compared to thepublished sequence of ULS to enable the selection of primers to be usedin the PCR amplification of the UL5 open reading frame. Restrictionsites were engineered into the 5′ ends of the primers for directionalcloning of the PCR product into a commercially available vector.

The PCR amplification of the ULS open reading frame yielded a 2.6 Kbfragment that was cloned into the pCRP™ 2.1 cloning vector (InvitrogenCorporation, Carlsbad, Calif.,). A 2.6 Kb HindIII-NotI fragment wassubcloned from the pCR™ 2.1 cloning vector into the constitutivemammalian expression vector pZeoSV2 (Invitrogen Corporation, Carlsbad,Calif.,). The pZeoSV2 expression vector contains the gene that confersresistance to the antibiotic Zeocin™ and the bovine growth hormone polyAsignal. The expression of the UL5 gene product is under the control ofthe SV40 early enhancer/promoter.

The resulting plasmid was designated p70-4.

EXAMPLE 4 Construction of an ICP8 Expressing Cell Line: VRA1

Vero Cells (African Green Monkey Kidney Cells, ATCC #81-CCL) were usedto create a cell line that expresses the ICP8 proteins of Herpes SimplexVirus by transfecting Vero cells with the HSV-1 ICP8 expression plasmid,pRC/CMV-ICP8-A1(RA1), described in example 2. A modified calciumphosphate method of transfection was used to create the ICP8 expressingcell line.

Four ampules of Vero Cells were removed from liquid nitrogen storage.The ampules were placed in a 37° C. water bath to rapidly thaw thecontents. The thawed cell suspensions were transferred to a sterile testtube containing chilled Dulbecco's Modified Eagle's Medium (DMEM). Thecells were harvested by centrifugation at 10,000 rpm for 10 minutes at40° C. in a Sorvall RT 6000D centrifuge. The supernatant was removedfrom the cell pellet and the cells were gently re-suspended in “V”media.

The cell suspension was transferred to a T25 cell culture flask. Thecells were fed and split through a series of passages using standardtissue cell culture procedures and materials known in the art. The cellswere harvested at each passage by dissociating the cells from theculture vessel with trypsin. The continuous culture and expansion of theVero cells was continued, resulting in a total of twenty two passages.Cell monolayers of the Vero cells at passage 22 (P22) were prepared in 6cm cell culture plates for transfection with the ICP8 expression plasmidpRC/CMV-ICP8-A1(RA1).

A solution containing 1.4 μg of plasmid DNA was prepared by adding 4 μlof the plasmid preparation (DNA concentration=0.35 μg/μl) to 259 μl ofsterile water in a sterile epindorf tube. A solution containing 8 μg ofplasmid DNA was prepared by adding 22.9 μl of the plasmid preparation(DNA concentration=0.35 μg/μl) to 240.1 μl of sterile water in a sterileepindorf tube. The contents of the tubes were mixed by vortexing. 37 μlof 2M CaCl₂ was added to each solution and mixed by vortexing.

300 μl of 2×HBS was added to two sterile polypropylene test tubes. Toprepare the DNA transfection solutions, the plasmid DNA/CaCl₂ solutionswere added dropwise to the 300 μl of 2×HBS. The polypropylene tubescontaining the 2×HBS were vortexed during the addition of the DNA/CaCl₂solutions. The DNA transfection solutions were incubated for 30 minutesat room temperature and then vortexed.

The solutions were separately added dropwise to two 6 cm plates of P22Vero cells. The plates containing the cell monolayers and plasmid DNAtransfection solutions were placed in a 37° C/5% CO₂ incubator for 24 to48 hours.

The cell culture plates containing the transfected Vero cells wereremoved from the incubator and the media was removed from the plate. Thecell monolayers were washed by adding 4 ml of PBS to each plate,swirling the plate for 1 minute and then decanting the PBS off theplate. 3 ml of trypsin was added to each plate, the plates were swirledto distribute the trypsin and the trypsin was poured off the plates. Theplates were placed in a 37° C./5% CO₂ incubator for 5-10 minutes.

V Media was added to ten (10), 10 cm cell culture plates (9.5 ml/plate).The plates containing the trypsinized cells were removed from theincubator and the plates were tapped to dislodge the cells from thebottom of the plate. 2.5 ml of V media was added to each plate. Thecells were re-suspended in the media with gentle repeat pipetting. There-suspended cells were transferred in 0.5 ml aliquots to the 10 cm cellculture plates containing 9.5 ml of V media. 100 μl of G418 solution(100 mg/ml) was added to each 10 cm cell culture plate. The plates wereincubated in a 37° C./5% CO₂ incubator for approximately four days.

The 10 cell culture plates containing the transfected Vero cells wereremoved from the incubator. The media was poured off the plates and 10ml of G media was added to each plate. The plates were returned to the37° C./5% CO₂ incubator for an additional 9 days.

Individual colonies of G418 resistant Vero cells were selected andtransferred to individual wells of 24-well cell culture plates. The 10cell culture plates containing the transfected Vero cells were removedfrom the incubator. The plates were vigorously swirled to dislodge anydead cells from the bottom of the plate. The media containing dead cellswas poured off the plates. The remaining cell colonies in each platewere washed with 5 ml of PBS. Individual cell colonies were visualizedand separately “picked”. Colonies were picked by adding 2 μl of trypsindirectly to the colony. The cells were then gently scraped from theplate using a pipette tip. The cells were transferred into a well of a24-well cell culture plate containing 0.5 ml of G media. The 24-wellcell culture plates were placed in a 37° C./5% CO₂ incubator forapproximately 9 days.

The selected G418 resistant cells were transferred from the 24-well cellculture plates to 6-well cell culture plates. The 24-well cell cultureplates were removed from the incubator. The media was aspirated from thewells. 1 ml of Dulbecco's Modified Eagles Medium (DMEM) was added toeach well. The DMEM was aspirated from the wells after approximately 1minute of incubation. To each well 0.5 ml of trypsin was added. Theplates were swirled and the trypsin was aspirated from the wells. Theplates were incubated for 5-10 minutes in a 37° C./5% CO₂ incubator. Theplates were removed from the incubator and tapped to detach cells fromthe wells of the plate. The cells in each well were re-suspended in 1 mlof G media. The cell suspensions from individual wells were transferredto wells of a 6-well cell culture plate containing 1 ml of G media. The6-well plates were placed in a 37° C./5% CO₂ incubator for approximately4 days.

The selected G418 resistant cells were transferred from the 6-well cellculture plates to T25 cell culture flasks. The 6-well cell cultureplates were removed from the incubator. The media was aspirated from thewells. 2 ml of Dulbecco's Modified Eagles Medium (DMEM) was added toeach well. The DMEM was aspirated from the wells after approximately 1minute of incubation. To each well 0.5 ml of trypsin was added. Theplates were swirled and the trypsin was aspirated from the wells. Anadditional 200 μl of trypsin was added to each well. The plates weretapped to detach cells from the wells of the plate. The cells in eachwell were re-suspended in 1 ml of G media. The cell suspensions fromindividual wells were transferred to separate T25 cell culture flaskscontaining 5 ml of G media. The T25 flasks were placed in a 37° C./5%CO₂ incubator for approximately 6 days. The T25 cell culture flaskscontaining the selected G418 resistant cells were removed from theincubator. The media was removed from the flask and the cells werewashed with 6 ml of PBS. Trypsin was added to each flask (3 ml). Theflasks were placed on their sides for 30 seconds to allow the trypsin tobe distributed over the cell monolayer. The trypsin was decanted fromthe flask. Three (3) ml of G media was added to each flask and theflasks were tapped to dislodge the cells. Aliquots of the cellsuspensions were transferred to wells of 6-well cell culture platescontaining 1 ml of G media. From each T25 flask two, 1.25 ml aliquots ofthe cell suspension were transferred to individual wells of a 6-wellplate. The 6-well cell culture plates were prepared for the screening ofG418 resistant Vero cell clones for their ability to support replicationof the HSV-2 ΔICP8 Strain A1-1 (provided by David Knipe). The cellculture plates were placed in a 37° C./5% CO₂ incubator. 5.5 ml of Gmedia was added to each cell suspension remaining in the T25 flask (˜0.5ml) and the flasks were returned to the incubator.

EXAMPLE 4.1 Screening of ICP8 Expressing Cell Line VRA1 to Confirm G418Resistance

Vero cells transfected with the ICP8 expression vectorpRC/CMV-ICP8-A1(RA1), described in example 4, were grown in the presenceof G418 to select for transfected cells based on G418 resistanceconferred by the expression vector. The Vero cells transfected with asolution containing 8 μg of plasmid DNA yielded 27 individual G418resistant colonies that were selected and expanded.

EXAMPLE 4.2 Screening of ICP8 Expressing Cell Line VRA1 for the Abilityto Support Replication of HSV-2 ΔICP8 Strain A1-1

HSV-2 strain ΔICP8 A1-1 contains a mutation on the UL29 gene and doesnot express the ICP8 protein. A laboratory virus stock preparation ofHSV-2 strain ΔICP8 A1-1, passage 3 was removed from frozen storage. Theampule containing the viral stock was thawed in a 37° C. water bath andplaced on ice. The viral stock was diluted as follows:

Viral stock ΔICP8 A1-1 diluted 1:30,000:

-   -   Dilution #1: 50 μl viral stock+4.95 ml DPBS    -   Dilution #2: 50 μl Dilution #1+4.95 ml DPBS    -   Dilution #3: 2.0 ml Dilution #2+4.0 ml DPBS

VRA1 cell monolayers, as described earlier, were washed with 2 ml ofPBS. Each of six wells was inoculated with 200 μl of the diluted ΔICP8A1-1 virus preparation. In addition, cell monolayers of the ICP8expressing cell line JW73A (Avant Immunotherapeutics, Inc., Needham,Mass.) were inoculated with 200 μl of the diluted ΔICP8 A1-1 viruspreparation as a positive control for the plaque assay. The plates wereplaced in a 37° C./5% CO₂ incubator and rocked every ten minutes for 60minutes. 2 ml of Media 199 (Gibco Life Technologies, Rockville, Md.;including fetal calf serum, penicillin/streptomycin solution, gammaglobulin, and glutamine) was added to each well and the plates wereincubated for 2 days. The plates were removed from the incubator and thewells were observed for the presence of plaques.

Clones were scored as positive for ICP8 expression based on the numberand the size of resulting plaques, as compared to the positive controlcell line. A single clone (VRA1) was selected based on these criteria.The tissue culture plate wells containing the clone and the positivecontrol cell line JW73A had˜200-400 plaques of comparable size andmorphology.

The G418 resistant clone (VRA1), positive for its ability to supportreplication of HSV-2 ΔICP8 strain A1-1, was expanded and a frozen stockwas prepared. A T25 cell culture flask containing the VRA1 cell lineincubated for approximately 3 days. The media was removed from the flaskand the cell monolayer was washed with 6 ml of PBS. The cells weredetached with trypsin and re-suspended in 3 ml of G media. The cellsuspension was divided into three, 1 ml aliquots. Each 1 ml aliquot wastransferred to a T75 cell culture flask containing 12 ml of G media andincubated for approximately four days.

After incubation, media was poured from the flasks and the cellmonolayers were washed with PBS. The cells were detached with trypsinand re-suspended in G media. The cell suspension was divided into twoaliquots. Each aliquot was transferred to a T150 cell culture flaskcontaining G media. The T150 flasks were placed in a 37° C./5% CO₂incubator for an additional two days.

Following incubation, the media was removed from the flasks and the cellmonolayers were washed with PBS. The cells were detached with trypsinand re-suspended in 6 ml of DMEM containing 10% fetal bovine serum. TwoT75 cell culture flasks containing 12 ml of G media were inoculated with0.5 ml of the cell suspension. The flasks were placed in a 37° C./5% CO₂incubator. The remaining cell suspensions were transferred to a singlesterile polypropylene centrifuge tube and the volume was adjusted to 20ml with DMEM.

The cell suspension was centrifuged for 10 minutes at 4° C. at 1000 RPMin a Sorvall RT 6000D centrifuge. The supernatant was removed from thecell pellet and the cells were re-suspended in 2 ml of freezing media.To the 2 ml cell suspension a solution of DMEM containing 10% DMSO wasadded to a total volume of 4 ml. The cell suspension was slowly mixedwith a pipette and 1 ml aliquots were transferred to sterile cryovials.The cryovials were placed in a styrofoam box and were placed in a −20°freezer. The cryovials were transferred to vapor phase liquid nitrogenstorage the following day.

EXAMPLE 5 Construction of a UL5/ICP8 Expressing Cell Line: V295

The ICP8 expressing VRA1 cell line from example 4.2 was transfected withthe UL5 expression plasmid p70-4 (described in example 3) byelectroporation.

Cell monolayers of VRA1 were grown in T150 flasks. The cell monolayerswere washed with 25 ml PBS. The cell monolayers were coated with trypsinand incubated for 30 seconds at room temperature. The cells weredetached from the flask surface by tapping the flasks. The cells in eachflask were re-suspended with 6 ml of S2 media.

Cell suspensions were combined in a single sterile polypropylenecentrifuge tube. A 100 μl sample of the cell suspension was added to atest tube containing 900 μl PBS (1:10 dilution). The cell suspension wasdiluted 1:2 with trypan blue dye and the cell density was determinedusing a hemacytometer. The cell density was determined to be 1.9×10⁶cells/ml. The total number of cells in the cell suspension wascalculated to be 2.3×10⁷. The cell suspension was centrifuged for 10minutes at 4° C. at 1000 RPM in a Sorvall RT 6000D centrifuge. Thesupernatant was removed from the cell pellet and the cells werere-suspended in 0.46 ml of 1×HBS (˜5×10⁷ cells /ml).

A mixture of VRA1 cells and p70-4 plasmid DNA was prepared forelectroporation. A 200 μl sample of the cell suspension was transferredto an epindorf tube. 3 μl of laboratory stock plasmid p70-4 was added tothe cell suspension. 12 μl of 10% dextrose was added to the tube and thevolume was adjusted to 800 μl using 1× HBS. The cell suspension wasmixed and transferred to an electroporation cuvette.

The electroporator was set at 960 μFd and 0.25 volts. The cuvette wasplaced in the electroporator and pulsed as per instructions located inthe electroporation apparatus operating manual. Followingelectroporation, the cuvette was placed on ice. The contents of theelectroporation cuvette were transferred to a 10 cm cell culture platecontaining 10 ml V media. The plate was placed in a 37° C./5% CO₂incubator for approximately 2 days.

After incubation, the cell culture plate containing the electroporatedVRA1 cell monolayer was washed by adding 10 ml of PBS to the plate,swirling the plate for 1 minute and then decanting the PBS off theplate. Five (5) ml of trypsin was added to the plate, the plate wasswirled to distribute the trypsin and the trypsin was poured off theplate. An additional 500 μl of trypsin was added to the plate. S2Z Mediawas added to ten, 10 cm cell culture plates (9.5 ml/plate). The platescontaining the trypsinized cells were removed from the incubator and theplates were tapped to dislodge the cells from the bottom of the plate.5.0 ml of S2Z media was added to each plate. The cells were re-suspendedin the media with gentle repeat pipetting. The re-suspended cells weretransferred in 0.5 ml aliquots to the 10 cm cell culture platescontaining 9.5 ml of S2Z media. The plates were incubated in a 37° C./5%CO₂ incubator for an additional 12 days.

Following incubation, individual colonies of Zeocin resistant VRA1 cellswere selected and transferred to individual wells of 24 well cellculture plates. The 10 cell culture plates containing the transfectedVero cells were removed from the incubator. The plates were vigorouslyswirled to dislodge any dead cells from the bottom of the plate. Themedia containing dead cells was poured off the plates. The remainingcell colonies in each plate were washed with 5 ml of PBS. Individualcell colonies were visualized and separately “picked”. Colonies werepicked by adding 3 μl of trypsin directly to the colony. The cells werethen gently scraped from the plate using a pipette tip. The cells weretransferred into a well of a 24-well cell culture plate containing 0.5ml of S2Z media. The 24-well cell culture plates were placed in a 37°C./5% CO₂ incubator for approximately six days.

The selected Zeocin resistant VRA1 cells were transferred from the24-well cell culture plates to 6-well cell culture plates. The 24-wellcell culture plates were removed from the incubator. The media wasaspirated from the wells. 1 ml of Dulbecco's Modified Eagles Medium(DMEM) was added to each well. The DMEM was aspirated from the wellsafter approximately 1 minute of incubation. To each well 0.5 ml oftrypsin was added. The plates were swirled and the trypsin was aspiratedfrom the wells. The plates were incubated for 5-10 minutes in a 37°C./5% CO₂ incubator. The plates were removed from the incubator andtapped to detach cells from the wells of the plate. The cells in eachwell were re-suspended in 1 ml of S2Z media. The cell suspensions fromindividual wells were transferred to wells of a 6-well cell cultureplate containing 1 ml of S2Z media. The 6-well plates were placed in a37° C./5% CO₂ incubator for approximately six days.

The selected Zeocin resistant VRA1 cells were transferred from the6-well cell culture plates to T25 cell culture flasks. The 6-well cellculture plates were removed from the incubator. The media was aspiratedfrom the wells. 2 ml of Dulbecco's Modified Eagles Medium (DMEM) wasadded to each well. The DMEM was aspirated from the wells afterapproximately 1 minute of incubation. To each well 0.5 ml of trypsin wasadded. The plates were swirled and the typsin was aspirated from thewells. An additional 200 μl of trypsin was added to each well. Theplates were tapped to detach cells from the wells of the plate. Thecells in each well were re-suspended in 1 ml of G media. The cellsuspensions from individual wells were transferred to separate T25 cellculture flasks containing 5 ml of S2Z media. The T25 flasks were placedin a 37° C./5% CO₂ incubator for approximately 4 days.

The T25 cell culture flasks containing the selected G418 resistant cellswere removed from the incubator. The media was removed from the flasksand the cells were washed with 6 ml of PBS. Trypsin was added to eachflask (3 ml). The flasks were placed on their sides for 30 seconds toallow the trypsin to be distributed over the cell monolayer. The trypsinwas decanted from the flask. 3 ml of S2Z media was added to each flaskand the flasks were tapped to dislodge the cells. Aliquots of the cellsuspensions were transferred to wells of 6-well cell culture platescontaining 1 ml of S2Z media. From each T25 flask two, 1.25 ml aliquotsof the cell suspension were transferred to individual wells of a 6-wellplate.

The 6-well cell culture plates were prepared for the screening of Zeocinresistant VRA1 cell clones for their ability to support replication ofthe HSV-2 ΔICP8 Strain A1-1 and the HSV-2 ΔUL5 Strain Hr99 (provided byDavid Knipe). The cell culture plates were placed in a 37° C./5% CO₂incubator. 5.5 ml of S2Z media was added to each cell suspensionremaining in the T25 flask (˜0.5 ml) and the flasks returned to theincubator.

EXAMPLE 5.1 Screening of UL5/ICP8 Expressing Cell Line V295 to ConfirmG418/Zeocin Resistance

VRA1 cells electroporated with the UL5 expression vector p70-4 weregrown in the presence of the antibiotics G418 and Zeocin to select forcells bearing both of the expression vectors. Of the G418/Zeocinresistant colonies that arose following passage of the transfected cellsin media containing G418 and Zeocin, 150 colonies were selected andexpanded.

EXAMPLE 5.2 Screening of UL5/ICP8 Expressing Cell Lien V295 for theAbility to Support Replication of HSV-2 ΔICP8 Strain A1-1 and HSV-2 ΔUL5Strain HR99

The ICP8/UL5 expressing V295 cell line was selected on the basis of it'sability to support the replication of both an ICP8 mutant HSV-2 strain,and a UL5 mutant HSV-2 strain and a frozen stock prepared.

HSV-2 strain ΔICP8 A1-1 contains a mutation on the UL29 gene and doesnot express the ICP8 protein. HSV-2 strain ΔUL5 Hr99 contains a mutationin the UL5 gene and does not express the UL5 protein. Laboratory virusstock preparations of HSV-2 strain ΔICP8 A1-1, passage 2 and HSV-2strain ΔUL5 Hr99 were removed from frozen storage. Ampules containingthe viral stocks were thawed in a 37° C. water bath and placed on ice.The viral stocks were diluted as follows:

HSV-2 strain ΔICP8 A1-1 P2 stock (titer=4.6×10⁷ pfu/ml) diluted1:23,000:

-   -   Dilution #1 (1: 1000): 5 μl viral stock+5.0 ml DPBS    -   Dilution #2 (1:23): 625 μl Dilution #1+14.3 ml DPBS

HSV-2 strain ΔUL5 Hr99 stock (titer=1.6×10⁸ pfu/ml) diluted 1:80,000:

-   -   Dilution #1 (1:1000): 5 μl viral stock+5.0 ml DPBS    -   Dilution #2 (1:80): 188 μl Dilution #1+14.8 ml DPBS

Zeocin/G418 resistant VRA1 cell monolayers, as described earlier, werewashed with 2 ml of PBS. Each of six wells was inoculated with either a)200 μl of the diluted ΔICP8 A1-1 virus preparation (˜400 pfu), or b) 200μl of the diluted ΔUL5 Hr99 virus preparation (˜400 pfu). In addition,cell monolayers of the ICP8 expressing cell line JW73A and the UL5expressing cell line L2-5 (obtained from Dr. David Knipe, HarvardMedical School) were each inoculated with 200 μl of their respectivediluted virus preparation as positive controls for the plaque assay. Theplates were placed in a 37° C./5% CO₂ incubator and rocked every tenminutes for 60 minutes. 2 ml of Media 199-0 was added to each well andthe plates were incubated for 4 days. The plates were removed from theincubator and the wells were observed for the presence of plaques.

Clones were scored as positive for ICP8 and UL5 expression based on thenumber and the size of resulting plaques, as compared to the positivecontrol cell lines. A single clone (V295) was selected based on thesecriteria. The results of the plaque assay for the V295 isolate are asfollows:

Infected Infected Negative with HSV-2 with HSV-2 Infection Cell LineΔICP8 Strain A1-1 ΔUL5 Strain Hr99 Control (PBS) JW73A ˜200 plaques    0plaques 0 plaques L2-5    0 plaques ˜200 plaques 0 plaques V295 ˜200plaques ˜200 plaques 0 plaques

A Zeocin/G418 resistant cell line (V295, ATCC deposit PTA-2403),positive for its ability to support replication of HSV-2 ΔICP8 strainA1-1 and HSV-2 ΔUL5 strain Hr99 was expanded. The cell line was expandedthrough a series of passages using standard cell culturing methods. Totransfer cells to new culture vessels at each passage, the culture mediawas removed from the culture vessel, the cells were washed with PBS andthe cells were dissociated from the vessel using trypsin. Cellsuspensions were transferred to new culture vessels containing anappropriate amount of S2Z culture media.

T150 cell culture flasks containing final passage (P10) ICP8/UL5expressing cell line V295 were removed from the incubator. The media wasremoved from the flasks and the cell monolayers were washed with PBS.The cells were detached with trypsin and re-suspended in 5 ml of DMEMcontaining 10% fetal bovine serum. The cell suspensions were transferredto a single sterile polypropylene centrifuge tube and the volume wasadjusted to 20 ml with DMEM.

The cell suspension was centrifuged for 10 minutes at 4° C. at 1000 RPMin a Sorvall RT 6000D centrifuge. The supernatant was removed from thecell pellet and the cells were re-suspended in 2 ml of freezing media. 4ml of a solution of DMEM containing 10% DMSO was added to the cellsuspension. The cell suspension was slowly mixed with a pipette and six,1 ml aliquots were transferred to sterile cryovials. Four cryovialscontaining 0.5 ml of the cell suspension were prepared using theremaining 2 ml of cell suspension. The cryovials were placed in astyrofoam box and were placed in a −80° freezer. The cryovials weretransferred to vapor phase liquid nitrogen storage the following day.

EXAMPLE 6 Sequence Homology between a Replication-defective HSV-2 Mutantand the UL5/ICP8 Expressing Cell Line

The amount of DNA sequence homology between the UL5/ICP8 expressing cellline and the UL5/ICP8 double mutant virus, was analyzed by consideringeach deletion in the virus separately, and in the context of thecorresponding genetic construct used to create the complementing cellline (UL5 and ICP8 expression vectors).

Genetic information used in assessing the mutations created in thedouble mutant HSV-2 vaccine strain was provided by Xavier De Costa andDavid Knipe. Two single, UL5 and ICP8, mutants of HSV-2 were constructedin Dr. Knipe's laboratory. Viral DNA from the ICP8 and UL5 defectiveviruses was co-transfected into a cell line capable of complementingboth defects to isolate a virus defective in both UL5 and ICP8.Information about the constructed deletions in the UL5 and ICP8 deletedvirus was obtained from sequencing data provided by David Knipe (HarvardMedical School).

EXAMPLE 6.1 Confirming Minimal DNA Sequence Homology between a UL5Defective HSV-2 and the UL5/ICP8 Expressing Cell Line

The 2900 bp deletion in UL5 ORF in the recombinant virus starts 104 bpinto the ORF at the 5′ end and extends 361 bp past the stop codon. Adeletion of the first hundred nucleotides of the UL5 ORF would have alsocaused a deletion in the overlapping UL6 ORF. This 104 bp regionrepresents the only region of sequence homology between thecomplementing cell line and the ICP8/UL5 double mutant virus.

EXAMPLE 6.2 Confirming Minimal DNA Sequence Homology between an ICP8Defective HSV-2 and the UL5/ICP8 Expressing Cell Line

The 3743 bp deletion of the HSV-2 ICP8 gene begins approximately 82 bpupstream of the 5′ end of the ICP8 ORF and extends past thetranslational stop codon of ICP8 in the recombinant virus. There is nosequence homology between the UL29 sequence present in the complementingcell line and the ICP8/UL5 double mutant virus.

1. A method for producing a replication-defective, ICP8/UL5-defectiveherpesvirus double mutant comprising the steps of: (a) propagating saidICP8/UL5-defective herpesvirus using a cell line that is able to supportthe growth said virus; and (b) harvesting the virus resulting from step(a).
 2. The method of claim 1, wherein said replication-defectiveICP8/UL5-defective herpesvirus double mutant is a replication-defectiveΔICP8/ΔUL5 HSV-2 strain.
 3. The method of claim 2, wherein said cellline is a UL5/ICP8 expressing cell line.
 4. The method of claim 3,wherein said cell line is a Vero cell line.
 5. The method of claim 4,wherein said cell line exhibits the expression characteristics of cellline V295 as deposited with the ATCC and assigned deposit designationPTA-2403.
 6. A method for producing an HSV-2 vaccine comprising themethod of claim 5, and further comprising the step: (c) preparing avaccine from the harvested virus of step (b).
 7. A complementing cellline, wherein said cell line is able to support the propagation of areplication-defective, ICP8/UL5-defective HSV double mutant.
 8. The cellline of claim 7, wherein said replication-defective, ICP8/UL5-defectiveHSV double mutant is an ICP8/UL5-defective HSV-2 strain.
 9. The cellline of claim 8, wherein said ICP8/UL5-defective HSV-2 strain is aΔICP8/ΔUL5 HSV-2 strain.
 10. The cell line of claim 8, wherein said cellline is a UL5/ICP8 expressing cell line.
 11. The cell line of claims 10,wherein said cell line exhibits the expression characteristics of cellline V295 as deposited with the ATCC and assigned deposit designationPTA-2403.
 12. The cell line of claim 10 containing an ICP8/UL5-defectiveHSV-2 strain.
 13. The cell line of claim 11 containing anICP8/UL5-defective HSV-2 strain.